SUPPLEMENTARY INFORMATION · 2015-09-23 · SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124 ... Hlan On (KHO) in the QTL region, while selecting against the rest of the genome

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.124

NATURE PLANTS | www.nature.com/natureplants 1

1

A trehalose-6-phosphate phosphatase enhances anaerobic germination

tolerance in rice

Tobias Kretzschmar, Margaret Anne F. Pelayo, Kurniawan R. Trijatmiko, Lourd F. Gabunada,

Rejbana Alam, Rosario Jimenez, Merlyn S. Mendioro, Inez H. Slamet-Loedin,

Nese Sreenivasulu, Julia Bailey-Serres, Abdelbagi M. Ismail, David J. Mackill,

Endang M. Septiningsih

Supplementary Information

http://dx.doi.org/10.1038/nplants.2015.124

2 NATURE PLANTS | www.nature.com/natureplants

SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124

2

Supplementary Methods

Near isogenic line (NIL) development to fine map the qAG-9-2

NILs were developed for the quantitative trait locus (QTL), qAG-9-2 by backcrossing selected

BC2F3 progenies to the recurrent parent IR647, and maintaining a small introgression from Khao

Hlan On (KHO) in the QTL region, while selecting against the rest of the genome with

background DNA markers. Seven BC4F3 introgression lines developed from two BC2F3 from the

original mapping populations were used to confirm the presence of the QTL. Selected BC4F4

recombinant families were used to fine map qAG-9-2 (Primers # 1-6, 73-74; SSR markers

RM3769, RM24141, RM24161, RM105), first using 260 lines from 38 different NILs with

overlapping introgressions. Genotyping was performed to identify new recombinants, which

were then phenotyped and further genotyped along with the informative recombinants identified

in the first batch using additional markers (Primers #7-72). Fifty recombinants from nine

different NILs aided further fine mapping (Supplementary Fig.1). NIL-AG1 (IR 93312-30-101-

20-3-66-6) had the smallest introgression at qAG-9-2 and was confirmed to be 99% similar to

IR64 across 4,037 single nucleotide polymorphism (SAG1-) markers using a Rice 6K Illumina

Infinium assay designed by S. McCouch (Cornell University) and run at the Genotyping Services

Laboratory, IRRI (http://gsl.irri.org/). A single SAG1- at 12.326 Mb near qAG-9-2 on

chromosome 9 was polymorphic between IR64 and KHO. NIL66 possesses the KHO SAG1-

(Supplementary Fig. 8).

3

Survival rate experiment

Survival rate experiment under anaerobic germination (AG) in the greenhouse was conducted

following an established protocol6. For the QTL confirmation, 16 trays per replication were used

to accommodate 144 BC4F3 introgression lines and the two parental controls in each tray, thus a

total of 176 entries were used per replication. Seedling survival was scored 21 days after sowing

for two replicates. Randomization for all entries including the parental controls was performed

with Alpha Plus design. Germination was also performed in air on moistened paper towels.

Germination after 7 days was scored. Phenotypic screening for fine mapping was performed with

a similar set up.

De novo-assembly of qAG-9-2 from KHO

A Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with the

Arizona Genomics Institute (AGI). Established methods were used for BAC library

construction36, 41 with high molecular weight (HMW) nuclear DNA cleaved with HindIII (BAC

library OSIKBa available from AGI Resource Center (http://www.genome.arizona.edu/orders/).

BAC clones were spotted onto Hybond Filters using a Genetix Qbot and processed and

hybridized with 32P labeled probes “according to manufacturer’s instructions” (“ATMI”).

TheKHO_360 probe was generated by amplification of KHO genomic DNA in the Os09g20360

region using AG1-360_F and AG1-360_R (#75-76) and cloning of the ~1.4 kb XhoI and SpeI

fragment in pGEM-T Easy (Promega). The KHO_390 probe was obtained via digestion of the

KHO_Os09g20390 clone in pCR4Blunt-TOPO plasmid with PvuI, resulting in a ~1.2 kb

fragment (for cloning of this fragment see below). Probe labeling with 32P was by use of the

DECAprime II kit (Ambion, part no. AM1455) “ATMI”. Hybridization was carried out as




2

Supplementary Methods

Near isogenic line (NIL) development to fine map the qAG-9-2

NILs were developed for the quantitative trait locus (QTL), qAG-9-2 by backcrossing selected

BC2F3 progenies to the recurrent parent IR647, and maintaining a small introgression from Khao

Hlan On (KHO) in the QTL region, while selecting against the rest of the genome with

background DNA markers. Seven BC4F3 introgression lines developed from two BC2F3 from the

original mapping populations were used to confirm the presence of the QTL. Selected BC4F4

recombinant families were used to fine map qAG-9-2 (Primers # 1-6, 73-74; SSR markers

RM3769, RM24141, RM24161, RM105), first using 260 lines from 38 different NILs with

overlapping introgressions. Genotyping was performed to identify new recombinants, which

were then phenotyped and further genotyped along with the informative recombinants identified

in the first batch using additional markers (Primers #7-72). Fifty recombinants from nine

different NILs aided further fine mapping (Supplementary Fig.1). NIL-AG1 (IR 93312-30-101-

20-3-66-6) had the smallest introgression at qAG-9-2 and was confirmed to be 99% similar to

IR64 across 4,037 single nucleotide polymorphism (SAG1-) markers using a Rice 6K Illumina

Infinium assay designed by S. McCouch (Cornell University) and run at the Genotyping Services

Laboratory, IRRI (http://gsl.irri.org/). A single SAG1- at 12.326 Mb near qAG-9-2 on

chromosome 9 was polymorphic between IR64 and KHO. NIL66 possesses the KHO SAG1-

(Supplementary Fig. 8).

3

Survival rate experiment

Survival rate experiment under anaerobic germination (AG) in the greenhouse was conducted

following an established protocol6. For the QTL confirmation, 16 trays per replication were used

to accommodate 144 BC4F3 introgression lines and the two parental controls in each tray, thus a

total of 176 entries were used per replication. Seedling survival was scored 21 days after sowing

for two replicates. Randomization for all entries including the parental controls was performed

with Alpha Plus design. Germination was also performed in air on moistened paper towels.

Germination after 7 days was scored. Phenotypic screening for fine mapping was performed with

a similar set up.

De novo-assembly of qAG-9-2 from KHO

A Bacterial Artificial Chromosome (BAC) library was constructed in collaboration with the

Arizona Genomics Institute (AGI). Established methods were used for BAC library

construction36, 41 with high molecular weight (HMW) nuclear DNA cleaved with HindIII (BAC

library OSIKBa available from AGI Resource Center (http://www.genome.arizona.edu/orders/).

BAC clones were spotted onto Hybond Filters using a Genetix Qbot and processed and

hybridized with 32P labeled probes “according to manufacturer’s instructions” (“ATMI”).

TheKHO_360 probe was generated by amplification of KHO genomic DNA in the Os09g20360

region using AG1-360_F and AG1-360_R (#75-76) and cloning of the ~1.4 kb XhoI and SpeI

fragment in pGEM-T Easy (Promega). The KHO_390 probe was obtained via digestion of the

KHO_Os09g20390 clone in pCR4Blunt-TOPO plasmid with PvuI, resulting in a ~1.2 kb

fragment (for cloning of this fragment see below). Probe labeling with 32P was by use of the

DECAprime II kit (Ambion, part no. AM1455) “ATMI”. Hybridization was carried out as




4

previously described42, followed by visualization with a STORM PhosphorImager “ATMI”.

Positive BAC clones were gathered from the arrayed library and confirmed by secondary

hybridization and PCR. One BAC clone (BAC4), positive for both probes, was sequenced on a

Ion Personal Genome Machine (PGM) (Life Technologies) resulting in a total of 310,000 reads

with an average length of 255 bp. Reads were trimmed, filtered for BAC vector sequence and the

rice genomic sequence was assembled using DNASTAR 11 SeqMan NGen (Lasergene). The

assembly covered chromosome 9 from LOC_Os09g20340 to LOC_Os09g20390. As the region

LOC_Os09g20390 to LOC_Os09g20400, not covered by BAC4, this portion of the qAG-9-2

region was assembled via cloning and Sanger-based sequencing of overlapping amplicons.

Fragments were amplified with primer pairs AMP5.4 to AMP5.8 (#77-86), AG400_B (#87-88),

and AG1-400 (#89-90). Resulting products were cloned into the pGEM-T Easy (Promega) and

sequenced by a service provider (Macrogen) with their in-house M13_F and M13_R primers and

amplicon-specific primers spaced at a distance of ~500 bp along the expected sequence (primers

#91-115) and sequences assembled using DNASTAR 11 SeqMan Pro (Lasergene). The final

KHO qAG-9-2 region was assembled and annotated manually in DNASTAR 11 SeqBuilder

(Lasergene). Alignment of qAG-9-2 from KHO, Nipponbare (MSU7) and the IR64 assembly11

was performed via the MAUVE algorithm in DNASTAR 11 MegAlign Pro (Lasergene).

The 20 kb deletion of IR64 in qAG-9-2 was confirmed via amplification of the deletion flanking

region with primers DFR_F2 and DFR_R2 (#159-160). The resulting ~450 bp fragment was

cloned into pGEM-T Easy and sequenced (Macrogen). DFR_F2 and DFR_R2 in combination

with DFR_LB2 were used as co-dominant markers to track presence/absence of the deletion in

rice germplasm (Supplementary Fig. 2c).

5

Cloning, transformation and genotyping of the mutant plants

The full-length coding region of OsTPP7 (LOC_Os09g20390) was amplified from genomic

KHO DNA using TPP_F and TPP_R (#116-117), which introduced AvrII and KpnI restriction

sites. prUbi-F and prUbi-R (#118-119) were used to amplify a 1986 bp fragment of the maize

polyubiquitin promoter from pCAMBIA1300int::pZmUbi::tNOS (provided by Emmanuel

Guiderdoni, CIRAD, France) and introduced with HindIII and AvrII restriction sites.

TPP_P_Hind_F (#120) and TPP_P_AvrII_R (#121) or TPP_P_BamHI_R (#122) were used to

amplify a 1927 bp fragment of the OsTPP7 promoter from genomic KHO DNA. GUS_BamHI_F

and GUS_KpnI_R (#123-124) were used to amplify a 2053 bp fragment of the GUS reporter

gene from pCAMBIA1301 (CAMBIA, Brisbane, Queensland, Australia). The fragments were

introduced into the pCR4Blunt-TOPO vector (Invitrogen) “ATMI”. The overexpression

construct pZmUbi::OsTPP7 (OX) was assembled by ligating the maize polyubiquitin promoter

(HindIII-AvrII fragment) and OsTPP7 (AvrII-KpnI fragment) into the HindIII / KpnI cleaved

pCAMBIA1300int::tNOS. The native promoter construct pOsTPP7::OsTPP7 (AG1-) was

assembled by replacement of the pZmUbi fragment with the pOsTPP7 fragment using HindIII

and AvrII restriction sites. The pOsTPP7::GUS reporter gene construct was assembled by

inserting the TPP_P promoter fragment (1927 bp) and the GUS gene fragment (2053 bp) into the

HindIII / KpnI cleaved pCAMBIA1300int::tNOS.

Agrobacterium-mediated transformation of pZmUbi::OsTPP7 and pOsTPP7::OsTPP7 into IR64

was performed using immature embryos38. pOsTPP7::GUS was transformed into KHO using

calli derived from mature seeds38. The Agrobacterium strain LBA4404 was employed.

Regenerated transgenic plantlets (T0) were transferred to the greenhouse, grown in hydroponic

culture and transplanted into potted soil after three weeks.




4

previously described42, followed by visualization with a STORM PhosphorImager “ATMI”.

Positive BAC clones were gathered from the arrayed library and confirmed by secondary

hybridization and PCR. One BAC clone (BAC4), positive for both probes, was sequenced on a

Ion Personal Genome Machine (PGM) (Life Technologies) resulting in a total of 310,000 reads

with an average length of 255 bp. Reads were trimmed, filtered for BAC vector sequence and the

rice genomic sequence was assembled using DNASTAR 11 SeqMan NGen (Lasergene). The

assembly covered chromosome 9 from LOC_Os09g20340 to LOC_Os09g20390. As the region

LOC_Os09g20390 to LOC_Os09g20400, not covered by BAC4, this portion of the qAG-9-2

region was assembled via cloning and Sanger-based sequencing of overlapping amplicons.

Fragments were amplified with primer pairs AMP5.4 to AMP5.8 (#77-86), AG400_B (#87-88),

and AG1-400 (#89-90). Resulting products were cloned into the pGEM-T Easy (Promega) and

sequenced by a service provider (Macrogen) with their in-house M13_F and M13_R primers and

amplicon-specific primers spaced at a distance of ~500 bp along the expected sequence (primers

#91-115) and sequences assembled using DNASTAR 11 SeqMan Pro (Lasergene). The final

KHO qAG-9-2 region was assembled and annotated manually in DNASTAR 11 SeqBuilder

(Lasergene). Alignment of qAG-9-2 from KHO, Nipponbare (MSU7) and the IR64 assembly11

was performed via the MAUVE algorithm in DNASTAR 11 MegAlign Pro (Lasergene).

The 20 kb deletion of IR64 in qAG-9-2 was confirmed via amplification of the deletion flanking

region with primers DFR_F2 and DFR_R2 (#159-160). The resulting ~450 bp fragment was

cloned into pGEM-T Easy and sequenced (Macrogen). DFR_F2 and DFR_R2 in combination

with DFR_LB2 were used as co-dominant markers to track presence/absence of the deletion in

rice germplasm (Supplementary Fig. 2c).

5

Cloning, transformation and genotyping of the mutant plants

The full-length coding region of OsTPP7 (LOC_Os09g20390) was amplified from genomic

KHO DNA using TPP_F and TPP_R (#116-117), which introduced AvrII and KpnI restriction

sites. prUbi-F and prUbi-R (#118-119) were used to amplify a 1986 bp fragment of the maize

polyubiquitin promoter from pCAMBIA1300int::pZmUbi::tNOS (provided by Emmanuel

Guiderdoni, CIRAD, France) and introduced with HindIII and AvrII restriction sites.

TPP_P_Hind_F (#120) and TPP_P_AvrII_R (#121) or TPP_P_BamHI_R (#122) were used to

amplify a 1927 bp fragment of the OsTPP7 promoter from genomic KHO DNA. GUS_BamHI_F

and GUS_KpnI_R (#123-124) were used to amplify a 2053 bp fragment of the GUS reporter

gene from pCAMBIA1301 (CAMBIA, Brisbane, Queensland, Australia). The fragments were

introduced into the pCR4Blunt-TOPO vector (Invitrogen) “ATMI”. The overexpression

construct pZmUbi::OsTPP7 (OX) was assembled by ligating the maize polyubiquitin promoter

(HindIII-AvrII fragment) and OsTPP7 (AvrII-KpnI fragment) into the HindIII / KpnI cleaved

pCAMBIA1300int::tNOS. The native promoter construct pOsTPP7::OsTPP7 (AG1-) was

assembled by replacement of the pZmUbi fragment with the pOsTPP7 fragment using HindIII

and AvrII restriction sites. The pOsTPP7::GUS reporter gene construct was assembled by

inserting the TPP_P promoter fragment (1927 bp) and the GUS gene fragment (2053 bp) into the

HindIII / KpnI cleaved pCAMBIA1300int::tNOS.

Agrobacterium-mediated transformation of pZmUbi::OsTPP7 and pOsTPP7::OsTPP7 into IR64

was performed using immature embryos38. pOsTPP7::GUS was transformed into KHO using

calli derived from mature seeds38. The Agrobacterium strain LBA4404 was employed.

Regenerated transgenic plantlets (T0) were transferred to the greenhouse, grown in hydroponic

culture and transplanted into potted soil after three weeks.




6

T1 transformants were tested for T-DNA integration via PCR using HPT_F and HPT_R (#125-

126) that amplify a 600 bp fragment of the hygromycin resistance gene of the pCAMBIA1300

series. Homozygous transgenic plants and their wild type siblings were identified by germination

trials on hygromycin (75 µM) containing plates or a PCR scheme that recognized homozygosity.

T2 generation lines were furthermore checked for segregation of the antibiotic resistance trait.

Detailed studies were performed on two independent homozygous transgene insertion lines

(OX1 and OX2) out of 82 independent events generated with seed of the T2 and T3 generation.

Six independent pOsTPP7::OsTPP7 (AG1) lines were produced and TAIL PCRs43 performed on

the T1-derived genomic DNA using primers #127-133 to determine the T-DNA insertion sites

and allow for design of T-DNA flanking primers to determine homozygosity. Detailed studies

were performed on two independent homozygous lines (AG1-1 and AG1-2) of the T2 and T3

generations. The site of T-DNA insertion was outside of a known coding region of a gene in both

lines. Homozygosity for AG1-1 was evaluated by PCR using the insertion flanking AG1-

1_HOMO_F and AG1-1_HOMO_R primers (#134-135) together with the T-DNA left border

primer TDNA-LB4 (#128). Homozygosity for AG1-2 was determined using AG1-2_HOMO_F

and AG1-2_HOMO_R primers (#136-137) together with TDNA-LB4 (#130) (Supplementary

Fig. 4a-d).

For pOsTPP7::GUS plants 196 independent lines were generated, of which more than 50 were

tested positively for GUS staining in germinating tissues of the T1 generation. GUS staining was

performed as previously described44. Five independent lines in the T2 and T3 generation were

used for detailed studies.

A mutant with a T-DNA insertion in the third exon of LOC_Os09g20390 (Supplementary Fig.

4g) in the Dongjin background (CLON PFG_3A-08739.L) was obtained from the Crop

7

Developmental Biology Lab (http://cbi.khu.ac.kr). Seeds (T1) were germinated and the resulting

lines checked for homozygosity with a three primer approach, using the T-DNA insertion

flanking pfg_tpp_F and pfg_tpp_R (#138-139) and pfg_tpp_F in combination with the T_DNA

left border specific primer TLBP2 (#140) (Supplementary Fig. 4g-i). Homozygous mutant lines

and homozygous null segregant siblings were carried into the T2 and T3 generations and used

for phenotypic analysis. OsTPP7 expression was tested via semiquantitative RT-PCR (see

below) using KO_RT_F (#182) and KO_RT_R (#183).

Plant growth conditions for biochemical and molecular analyses

Seeds were stored at 4 °C after post-harvest processing. Seed dormancy was broken by

incubation at 50 °C For 5 days. Seeds were de-hulled, sterilized in 70% ethanol for 2 min,

washed three times with sterile water and submerged in 8 cm of autoclaved distilled water for 1-

4 days at 30 °C in the dark. AG was performed with at least three biological replicates, each with

≥ 23 individual coleoptiles per analysis. For germination in air, seeds were placed in sterile petri

dishes that contained moistened paper towels and kept for 1-4 days at 30 °C in the dark. For

sucrose treatment, water was substituted with sterile 90 mM sucrose (n=3, ≥ 46 coleoptiles per

analysis). Tissue was rapidly harvested and snapped frozen in liquid nitrogen and stored at -

80°C. For germination assays in the presence of hormones, seeds were germinated in the dark in

air at 23°C and germination was determined every 12 h (0.1-10 µM GA treatment) or 24 h (0.001

– 5 µM ABA treatment). GA treatment was in the presence of 200 µM paclobutrazol. Mock

treatments controlled for the quantity of DMSO used to solubilize hormones.




6

T1 transformants were tested for T-DNA integration via PCR using HPT_F and HPT_R (#125-

126) that amplify a 600 bp fragment of the hygromycin resistance gene of the pCAMBIA1300

series. Homozygous transgenic plants and their wild type siblings were identified by germination

trials on hygromycin (75 µM) containing plates or a PCR scheme that recognized homozygosity.

T2 generation lines were furthermore checked for segregation of the antibiotic resistance trait.

Detailed studies were performed on two independent homozygous transgene insertion lines

(OX1 and OX2) out of 82 independent events generated with seed of the T2 and T3 generation.

Six independent pOsTPP7::OsTPP7 (AG1) lines were produced and TAIL PCRs43 performed on

the T1-derived genomic DNA using primers #127-133 to determine the T-DNA insertion sites

and allow for design of T-DNA flanking primers to determine homozygosity. Detailed studies

were performed on two independent homozygous lines (AG1-1 and AG1-2) of the T2 and T3

generations. The site of T-DNA insertion was outside of a known coding region of a gene in both

lines. Homozygosity for AG1-1 was evaluated by PCR using the insertion flanking AG1-

1_HOMO_F and AG1-1_HOMO_R primers (#134-135) together with the T-DNA left border

primer TDNA-LB4 (#128). Homozygosity for AG1-2 was determined using AG1-2_HOMO_F

and AG1-2_HOMO_R primers (#136-137) together with TDNA-LB4 (#130) (Supplementary

Fig. 4a-d).

For pOsTPP7::GUS plants 196 independent lines were generated, of which more than 50 were

tested positively for GUS staining in germinating tissues of the T1 generation. GUS staining was

performed as previously described44. Five independent lines in the T2 and T3 generation were

used for detailed studies.

A mutant with a T-DNA insertion in the third exon of LOC_Os09g20390 (Supplementary Fig.

4g) in the Dongjin background (CLON PFG_3A-08739.L) was obtained from the Crop

7

Developmental Biology Lab (http://cbi.khu.ac.kr). Seeds (T1) were germinated and the resulting

lines checked for homozygosity with a three primer approach, using the T-DNA insertion

flanking pfg_tpp_F and pfg_tpp_R (#138-139) and pfg_tpp_F in combination with the T_DNA

left border specific primer TLBP2 (#140) (Supplementary Fig. 4g-i). Homozygous mutant lines

and homozygous null segregant siblings were carried into the T2 and T3 generations and used

for phenotypic analysis. OsTPP7 expression was tested via semiquantitative RT-PCR (see

below) using KO_RT_F (#182) and KO_RT_R (#183).

Plant growth conditions for biochemical and molecular analyses

Seeds were stored at 4 °C after post-harvest processing. Seed dormancy was broken by

incubation at 50 °C For 5 days. Seeds were de-hulled, sterilized in 70% ethanol for 2 min,

washed three times with sterile water and submerged in 8 cm of autoclaved distilled water for 1-

4 days at 30 °C in the dark. AG was performed with at least three biological replicates, each with

≥ 23 individual coleoptiles per analysis. For germination in air, seeds were placed in sterile petri

dishes that contained moistened paper towels and kept for 1-4 days at 30 °C in the dark. For

sucrose treatment, water was substituted with sterile 90 mM sucrose (n=3, ≥ 46 coleoptiles per

analysis). Tissue was rapidly harvested and snapped frozen in liquid nitrogen and stored at -

80°C. For germination assays in the presence of hormones, seeds were germinated in the dark in

air at 23°C and germination was determined every 12 h (0.1-10 µM GA treatment) or 24 h (0.001

– 5 µM ABA treatment). GA treatment was in the presence of 200 µM paclobutrazol. Mock

treatments controlled for the quantity of DMSO used to solubilize hormones.




8

RNA extraction, semiquantitative and quantitative RT-PCRs

Embryos and coleoptiles were dissected from seeds and ground in liquid nitrogen. RNA

extraction and clean-up was performed using the RNeasy kit (Qiagen) with an in-column DNAse

digest “ATMI”. Reverse transcription was performed using the GoScript Reverse transcription

system (Promega) “ATMI”. Semi-quantitative RT-PCR was performed for LOC_Os09g20360

(Primers #141-142), LOC_Os09g20370 (Primers #143-144), LOC_Os09g20390 (Primers #145-

146) and LOC_Os09g20400 (Primers #147-148), with α-tubulin (Primers #149-150) as

housekeeping control using 32 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, preceded

by 90 s of initial denaturation at 95 °C and followed by 90 s at 72 °C.

Quantitative RT-PCR was performed using a Lightcycler 480 (Roche) and Roche consumables

(“ATMI”) or MyiQ real-time PCR detection system (Bio-Rad) using iQ SYBR Green supermix

(Bio-Rad)45. Cycling parameters were: 45 cycles of 10 s at 95 °C, 15 s at 60°C and 8 s at 72 °C,

preceded by 5 s of initial denaturation at 95 °C. Primers pairs (#151-158 and 164-181) for genes

of interest and the references Tubulin, Polyubiquitin and Ubiquitin are in Supplementary Table 2.

For Supplementary Fig. 6a-c the REST software (Qiagen)46 set at 3000 iterations was used for

calculation of expression differences and statistical analysis across. For Supplementary Fig. 6d

relative transcript abundance across 4 biological replicates was calculated by the comparative CT

method47 and samples compared by ANOVA using R script. Primer efficiencies were

calculated by REST or manually via a dilution series and included in the analysis48.

9

Whole genome re-sequencing and RNA sequencing analysis

RNA was extracted using the RNeasy-Plant Mini kit (Qiagen) “ATMI”. RNA quality and

integrity was checked on a 2100 Bioanalyzer (Agilent) “ATMI”. Whole genome sequencing and

cDNA-based whole transcriptome sequencing (RNAseq) was performed by Macrogen on an

Illumina Hiseq2000 platform generating 100 bp paired-end reads with an average insert size of

300 bp. The generated Fastq files were processed and analyzed using the software suite CLC-

Genomics-Workbench 7 (Qiagen). All downstream analyses were performed within the CLC-

Genomics-Workbench 7 RNA Seq Analysis suite. EDGE-test was performed to generate fold-

differences and associated false discovery rate (FDR) corrected p-value and p-values.

Differentially expressed genes were compared to those reported in the apical 6mm tip and 5 mm

base of coleoptiles of rice cv. Amaroo seedlings germinated for 72 h in air or under 3% oxygen

(hypoxia)33 by K-means clustering49 and gene ontology50 analyses.

Analysis of T6P, trehalose and sucrose

Quantification of sucrose, trehalose-6-hosphate (T6P) and trehalose was performed by

Metabolomic Discoveries, Germany. Frozen embryo-coleoptile tissue was mechanically

disrupted in a ball mill in liquid nitrogen (Retsch), freeze dried, and 60 mg was mixed with 1 ml

80% (v/v) methanol and incubated for 15 min in a thermoshaker (1000 rpm) at 70 °C. Cellular

debris was removed by centrifugation. The extract supernatant was mixed with nine volumes of

90% (v/v) methanol and incubated for 15 min at 37 °C with vigorous shaking. Precipitated

protein was removed by centrifugation for 15 min at 13500 rpm and the supernatant retained.

Derivatisation and analyses of metabolites by a GC-MS 7890A mass spectrometer (Agilent)

were carried out as described40. Metabolites were identified in comparison to Metabolomic




8

RNA extraction, semiquantitative and quantitative RT-PCRs

Embryos and coleoptiles were dissected from seeds and ground in liquid nitrogen. RNA

extraction and clean-up was performed using the RNeasy kit (Qiagen) with an in-column DNAse

digest “ATMI”. Reverse transcription was performed using the GoScript Reverse transcription

system (Promega) “ATMI”. Semi-quantitative RT-PCR was performed for LOC_Os09g20360

(Primers #141-142), LOC_Os09g20370 (Primers #143-144), LOC_Os09g20390 (Primers #145-

146) and LOC_Os09g20400 (Primers #147-148), with α-tubulin (Primers #149-150) as

housekeeping control using 32 cycles of 30 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, preceded

by 90 s of initial denaturation at 95 °C and followed by 90 s at 72 °C.

Quantitative RT-PCR was performed using a Lightcycler 480 (Roche) and Roche consumables

(“ATMI”) or MyiQ real-time PCR detection system (Bio-Rad) using iQ SYBR Green supermix

(Bio-Rad)45. Cycling parameters were: 45 cycles of 10 s at 95 °C, 15 s at 60°C and 8 s at 72 °C,

preceded by 5 s of initial denaturation at 95 °C. Primers pairs (#151-158 and 164-181) for genes

of interest and the references Tubulin, Polyubiquitin and Ubiquitin are in Supplementary Table 2.

For Supplementary Fig. 6a-c the REST software (Qiagen)46 set at 3000 iterations was used for

calculation of expression differences and statistical analysis across. For Supplementary Fig. 6d

relative transcript abundance across 4 biological replicates was calculated by the comparative CT

method47 and samples compared by ANOVA using R script. Primer efficiencies were

calculated by REST or manually via a dilution series and included in the analysis48.

9

Whole genome re-sequencing and RNA sequencing analysis

RNA was extracted using the RNeasy-Plant Mini kit (Qiagen) “ATMI”. RNA quality and

integrity was checked on a 2100 Bioanalyzer (Agilent) “ATMI”. Whole genome sequencing and

cDNA-based whole transcriptome sequencing (RNAseq) was performed by Macrogen on an

Illumina Hiseq2000 platform generating 100 bp paired-end reads with an average insert size of

300 bp. The generated Fastq files were processed and analyzed using the software suite CLC-

Genomics-Workbench 7 (Qiagen). All downstream analyses were performed within the CLC-

Genomics-Workbench 7 RNA Seq Analysis suite. EDGE-test was performed to generate fold-

differences and associated false discovery rate (FDR) corrected p-value and p-values.

Differentially expressed genes were compared to those reported in the apical 6mm tip and 5 mm

base of coleoptiles of rice cv. Amaroo seedlings germinated for 72 h in air or under 3% oxygen

(hypoxia)33 by K-means clustering49 and gene ontology50 analyses.

Analysis of T6P, trehalose and sucrose

Quantification of sucrose, trehalose-6-hosphate (T6P) and trehalose was performed by

Metabolomic Discoveries, Germany. Frozen embryo-coleoptile tissue was mechanically

disrupted in a ball mill in liquid nitrogen (Retsch), freeze dried, and 60 mg was mixed with 1 ml

80% (v/v) methanol and incubated for 15 min in a thermoshaker (1000 rpm) at 70 °C. Cellular

debris was removed by centrifugation. The extract supernatant was mixed with nine volumes of

90% (v/v) methanol and incubated for 15 min at 37 °C with vigorous shaking. Precipitated

protein was removed by centrifugation for 15 min at 13500 rpm and the supernatant retained.

Derivatisation and analyses of metabolites by a GC-MS 7890A mass spectrometer (Agilent)

were carried out as described40. Metabolites were identified in comparison to Metabolomic




10

Discoveries database entries of authentic standards. The LC separation was performed using a

Zorbax SB-Aq column (Agilent), operated by an Agilent 1290 UPLC system (Agilent). The LC

mobile phase was A) 0.1% (v/v) formic acid in water and B) 0.1% (v/v) formic acid in methanol

with a gradient from 0% to 90% B over 5 min, to 95% at 6.5 min and 100% at 8 min, and

subsequently equilibrated. Flow rate was 400 µl/min, and injection volume 1 µl. Mass

spectrometry was performed using a 6540 QTOF/MS Detector (Agilent). Measured metabolite

concentration was normalized to internal standards. Significant concentration changes of

metabolites in different samples were analyzed by Student’s t-test. A p-value of p<0.05 was

considered significant. For absolute quantification, the amount of target metabolite was

compared to known concentrations of reference standards. Five biological replicates were used

for all analyses.

Analysis of monosaccharides, sugar alcohols and amino acids

Homogenized tissue (20 mg) was mixed with 100 µL decanoic acid (1 mg/mL in ethanol) and 1

mL hot ethanol. The mixture was extracted three times by incubation for 5 min in a 75 °C water

bath followed by centrifugation at 12,000 g, t he supernatants combined and dried in 2 mL

microfuge tubes using a SpeedVac concentrator. For derivatization, 135 µL BSTFA with 1%

(w/w) TMCS and 120 µL Pyridine was added, silanized at 75 °C for 1 h in a water bath,

centrifuged at 12,500 g for 30 sec and 1 µL was analyzed using an Agilent 6890 gas

chromatograph coupled to a 5975 MS (Agilent) using a DB-5MS (30 m x 250 µm x 0.25 µm)

column. The inlet was set to 280 °C and the oven at 60 °C for 4 min, ramped to 170 °C at 8

°C/min, then to 300 °C at 4°C/min and then maintained at 300 °C for 30 min. The carrier gas

was helium programmed to flow at 1.2mL/min.

11

The MS was set to scan from 50-650m/z in the electron impact mode at 70eV. The interface,

quadrupole and source were set at 285 °C, 150 °C and 230 °C respectively. Metabolites were

identified by comparison of the experimental mass spectrum to the NIST 11 MS Library

(Gaithersburg, MD, USA). Peak areas were normalized using the internal standard-corrected

method and the values were log transformed. Values were derived from 5 biological and 2

technical replicates. Fold change differences and statistical significance was assessed using the

Welch two-sample t-test with adjusted P-value for multiple corrections using Benjamini &

Hochberg. The relative amounts of the individual metabolites were represented in the form of a

heat map.

TPP enzyme production in E. coli, purification and activity assays

The TPP7 coding sequence (LOC_Os09g20390.1) was amplified from coleoptile cDNA using

the oligonucleotides FLEX_SV1_F and FLEX_SV1_R (#157-158) and cloned into pFN18A

Halo Tag T7 Flexi vector of the pFLEXI bacterial expression system (Promega) “ATMI”. The

construct was subsequently transformed into Single Step (KRX) competent E. coli cells

(Promega) “ATMI”. The recombinant TPP7 with a N-terminal Halo tag was induced in KRX

and purified using the HaloTag Protein Purification system (Promega) “ATMI”. TPP enzyme

activity was monitored spectrophotometrically by colorimetric quantification of released

phosphate from T6P as described previously21, using BIOMOL Green Reagent (Enzo Life

Science) and 0.2 µg purified TPP7 per assay. Substrate concentrations ranged from 0.1 to 1 mM

T6P.




10

Discoveries database entries of authentic standards. The LC separation was performed using a

Zorbax SB-Aq column (Agilent), operated by an Agilent 1290 UPLC system (Agilent). The LC

mobile phase was A) 0.1% (v/v) formic acid in water and B) 0.1% (v/v) formic acid in methanol

with a gradient from 0% to 90% B over 5 min, to 95% at 6.5 min and 100% at 8 min, and

subsequently equilibrated. Flow rate was 400 µl/min, and injection volume 1 µl. Mass

spectrometry was performed using a 6540 QTOF/MS Detector (Agilent). Measured metabolite

concentration was normalized to internal standards. Significant concentration changes of

metabolites in different samples were analyzed by Student’s t-test. A p-value of p<0.05 was

considered significant. For absolute quantification, the amount of target metabolite was

compared to known concentrations of reference standards. Five biological replicates were used

for all analyses.

Analysis of monosaccharides, sugar alcohols and amino acids

Homogenized tissue (20 mg) was mixed with 100 µL decanoic acid (1 mg/mL in ethanol) and 1

mL hot ethanol. The mixture was extracted three times by incubation for 5 min in a 75 °C water

bath followed by centrifugation at 12,000 g, t he supernatants combined and dried in 2 mL

microfuge tubes using a SpeedVac concentrator. For derivatization, 135 µL BSTFA with 1%

(w/w) TMCS and 120 µL Pyridine was added, silanized at 75 °C for 1 h in a water bath,

centrifuged at 12,500 g for 30 sec and 1 µL was analyzed using an Agilent 6890 gas

chromatograph coupled to a 5975 MS (Agilent) using a DB-5MS (30 m x 250 µm x 0.25 µm)

column. The inlet was set to 280 °C and the oven at 60 °C for 4 min, ramped to 170 °C at 8

°C/min, then to 300 °C at 4°C/min and then maintained at 300 °C for 30 min. The carrier gas

was helium programmed to flow at 1.2mL/min.

11

The MS was set to scan from 50-650m/z in the electron impact mode at 70eV. The interface,

quadrupole and source were set at 285 °C, 150 °C and 230 °C respectively. Metabolites were

identified by comparison of the experimental mass spectrum to the NIST 11 MS Library

(Gaithersburg, MD, USA). Peak areas were normalized using the internal standard-corrected

method and the values were log transformed. Values were derived from 5 biological and 2

technical replicates. Fold change differences and statistical significance was assessed using the

Welch two-sample t-test with adjusted P-value for multiple corrections using Benjamini &

Hochberg. The relative amounts of the individual metabolites were represented in the form of a

heat map.

TPP enzyme production in E. coli, purification and activity assays

The TPP7 coding sequence (LOC_Os09g20390.1) was amplified from coleoptile cDNA using

the oligonucleotides FLEX_SV1_F and FLEX_SV1_R (#157-158) and cloned into pFN18A

Halo Tag T7 Flexi vector of the pFLEXI bacterial expression system (Promega) “ATMI”. The

construct was subsequently transformed into Single Step (KRX) competent E. coli cells

(Promega) “ATMI”. The recombinant TPP7 with a N-terminal Halo tag was induced in KRX

and purified using the HaloTag Protein Purification system (Promega) “ATMI”. TPP enzyme

activity was monitored spectrophotometrically by colorimetric quantification of released

phosphate from T6P as described previously21, using BIOMOL Green Reagent (Enzo Life

Science) and 0.2 µg purified TPP7 per assay. Substrate concentrations ranged from 0.1 to 1 mM

T6P.




12

Statistical analysis

Normality of the data sets analyzed by t-tests was confirmed graphically by histograms.

For coleoptile (Fig. 1b-c and Supplementary Fig. 3c), shoot and root length measurements

(Supplementary Fig. 3e) “n” equals the total number of individual seedlings sampled per

experiment, pooled from three independent biological replications (individual beaker or petri

dish set-up). Seeds that had not germinated at all at the time of sampling were not included in the

analysis and hence the “n” differed between genotypes. The “n” displayed in the figure legends

therefore represents the minimum “n”. Mean and standard error of means (s.e.m.) are displayed,

with a two-tailed t-test analysis.

For amylase assays (Fig. 1d and Supplementary Fig. 3d) “n” equals the number of independent

biological replications (individual beaker set-up). Though normality of the data set could not be

confirmed due to small sample; size standard deviation (s.d.) for error display and two-tailed t-

test for statistical analysis was chosen.

For T6P-related metabolite assays (Fig. 2a-c) “n” equals the number of independent biological

replications (individual beaker set-up). Though skewness was present, two tailed t-tests were

chosen for statistical analysis.

For OsTPP7-catalyzed de-phosphorylation assays (Supplementary Fig. 5c) “n” equals the

number of independent biological replications. For error display the standard error of means

(s.e.m.) was chosen. For statistical analysis a two-tailed t-test was chosen.

For glucose sensitivity assays (Supplementary Fig. 5f) “n” equals the total number of individual

seedlings sampled for the experiment, which were pooled from three independent biological

13

replications (individual beaker or petri dish set-up). For error display the standard deviation (s.d.)

was chosen. For statistical analysis a two-tailed t-test was chosen.

For metabolomics (Fig. 2) and transcriptomics analysis (Fig. 3h) detailed description of the

statistical methodology are given in the respective sections above.




12

Statistical analysis

Normality of the data sets analyzed by t-tests was confirmed graphically by histograms.

For coleoptile (Fig. 1b-c and Supplementary Fig. 3c), shoot and root length measurements

(Supplementary Fig. 3e) “n” equals the total number of individual seedlings sampled per

experiment, pooled from three independent biological replications (individual beaker or petri

dish set-up). Seeds that had not germinated at all at the time of sampling were not included in the

analysis and hence the “n” differed between genotypes. The “n” displayed in the figure legends

therefore represents the minimum “n”. Mean and standard error of means (s.e.m.) are displayed,

with a two-tailed t-test analysis.

For amylase assays (Fig. 1d and Supplementary Fig. 3d) “n” equals the number of independent

biological replications (individual beaker set-up). Though normality of the data set could not be

confirmed due to small sample; size standard deviation (s.d.) for error display and two-tailed t-

test for statistical analysis was chosen.

For T6P-related metabolite assays (Fig. 2a-c) “n” equals the number of independent biological

replications (individual beaker set-up). Though skewness was present, two tailed t-tests were

chosen for statistical analysis.

For OsTPP7-catalyzed de-phosphorylation assays (Supplementary Fig. 5c) “n” equals the

number of independent biological replications. For error display the standard error of means

(s.e.m.) was chosen. For statistical analysis a two-tailed t-test was chosen.

For glucose sensitivity assays (Supplementary Fig. 5f) “n” equals the total number of individual

seedlings sampled for the experiment, which were pooled from three independent biological

13

replications (individual beaker or petri dish set-up). For error display the standard deviation (s.d.)

was chosen. For statistical analysis a two-tailed t-test was chosen.

For metabolomics (Fig. 2) and transcriptomics analysis (Fig. 3h) detailed description of the

statistical methodology are given in the respective sections above.




14

Supplementary Figures

a

b

RM5535RM8303 RM24433RM219 RM7175RM23911

RM3769 RM105RM5526

RM3769 RM105

RM3769 RM105RM24141 GST2bpGST6bp

Ann11bp RM24161PDC3u1

L LLLLHHHHHMLL

Survival

100.5 kb

804.8 kb

Ann11bp RM24161Drebdws4bp TP80-90_6UE400_G3

AG400_GC3

L

HHHL L

EP40_G1TPP_E3

TP80-90_1 Sdhups5bp

L

L

~50 kb

Survival

A B C DEF GHI J KL M

ND, E, O

J P

K-1 K-2 QA, IR64

61251411011321

#Family

#Family

310

11

16

12

~ 20 kb deleted in IR64

15

Supplementary Figure 1 | Fine mapping of qAG-9-2

qAG-9-2 was delimited to a region of ~50 kb using 260 BC4F4 lines from 38 recombinant

progenies with different sizes of introgression in the region of the QTL (A-M) a, Solid boxes

represent homozygous KHO introgression regions. Seedling survival of AG is represented by L

(Low: 1-19%), M (Medium: 28%) and H (high: 35-50%). b, A second round of fine mapping

using selected families (A, D, E, O, J, and K, which splits to K-1 and K-2) and additional 50

BC4F4 lines from 9 NIL-AG1s (N-Q) narrowed down qAG-9-2 to a region of 46.5 kb. Successive

dominant markers predicted a 20.9 kb deleted region in IR64.




14

Supplementary Figures

a

b

RM5535RM8303 RM24433RM219 RM7175RM23911

RM3769 RM105RM5526

RM3769 RM105

RM3769 RM105RM24141 GST2bpGST6bp

Ann11bp RM24161PDC3u1

L LLLLHHHHHMLL

Survival

100.5 kb

804.8 kb

Ann11bp RM24161Drebdws4bp TP80-90_6UE400_G3

AG400_GC3

L

HHHL L

EP40_G1TPP_E3

TP80-90_1 Sdhups5bp

L

L

~50 kb

Survival

A B C DEF GHI J KL M

ND, E, O

J P

K-1 K-2 QA, IR64

61251411011321

#Family

#Family

310

11

16

12

~ 20 kb deleted in IR64

15

Supplementary Figure 1 | Fine mapping of qAG-9-2

qAG-9-2 was delimited to a region of ~50 kb using 260 BC4F4 lines from 38 recombinant

progenies with different sizes of introgression in the region of the QTL (A-M) a, Solid boxes

represent homozygous KHO introgression regions. Seedling survival of AG is represented by L

(Low: 1-19%), M (Medium: 28%) and H (high: 35-50%). b, A second round of fine mapping

using selected families (A, D, E, O, J, and K, which splits to K-1 and K-2) and additional 50

BC4F4 lines from 9 NIL-AG1s (N-Q) narrowed down qAG-9-2 to a region of 46.5 kb. Successive

dominant markers predicted a 20.9 kb deleted region in IR64.




16

a

b

KHOqAG9-2region

10 kb

Os09g20360 Os09g20370 Os09g20380 Os09g20390 Os09g20400

BAC-based de novo assembly Sequenced amplicons

Drebdws4bp AG400_GC3OsTPP7

MSU7

KHO

IR64

A B C D E

A B C D E

bp

A=Os09g20360Expressed protein

B=Os09g20370Expressed protein

C=Os09g20380Transposon protein

D=Os09g20390 E=Os09g20400Expressed proteinOsTPP7

d

IR 6

IR26

IR 42

IR 64

IR74

PSB Rc 82

NSIC Rc 222

NSIC Rc 238

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INDEL22.2 Mb 22.8 Mb

c

Os09g20380 Os09g20400Os09g20390

DELETION

OsTPP7

17

Supplementary Figure 2 | Differences of qAG-9-2 region between KHO, IR64 and MSU7

and distribution of the OsTPP7 deletion across IRRI germplasm

a, Schematic of the qAG-9-2 region, assembled via a hybrid approach of Bacterial Artificial

Chromosome (BAC) deep sequencing and sequencing of overlapping amplicon clones. White

boxes on left and right indicate fine mapping flanking markers. Black boxes indicate gene

models (MSU ID) and grey arrows indicate direction of gene transcription. b, Mauve alignment

of the qAG-9-2 region from the Nipponbare reference assembly (MSU7), KHO and IR6411. First

row indicates size and relative position. Numbered rectangles indicate gene models (A-E =

LOC_Os09g20360 to LOC_Os09g20400 indicated in panel a). Green areas indicate well aligned

portions and grey areas indicate deletions. LOC_Os09g20390 encodes OsTPP7.

LOC_Os09g20380 encodes an unclassified transposable element that is partially deleted in IR64

and KHO. c, Schematic of the ~21 kb deletion region containing OsTPP7 as found in IR64.

Arrows indicate primer positions for co-dominant marker. Orange area indicates position within

deletion. d, Presence (white boxes) and absence (black boxes) of SNPseek37 core SNPs across

the larger qAG-9-2 region for 90 IRRI-derived varieties and breeding lines that are represented in

the 3000 genomes project12 (NSIC Rc222 and NSIC Rc238 are not present in SNPseek and were

added from IRRI-internal data). Each row represents a different genotype/breeding line (pink

lines indicating the subset named on the left) and each column a core SNP. The data shows

distribution of the OsTPP7-containing deletion across a representative range of IRRI-derived

cultivars including very early cultivars (IR6) and recently released varieties (NSIC Rc238).




16

a

b

KHOqAG9-2region

10 kb

Os09g20360 Os09g20370 Os09g20380 Os09g20390 Os09g20400

BAC-based de novo assembly Sequenced amplicons

Drebdws4bp AG400_GC3OsTPP7

MSU7

KHO

IR64

A B C D E

A B C D E

bp

A=Os09g20360Expressed protein

B=Os09g20370Expressed protein

C=Os09g20380Transposon protein

D=Os09g20390 E=Os09g20400Expressed proteinOsTPP7

d

IR 6

IR26

IR 42

IR 64

IR74

PSB Rc 82

NSIC Rc 222

NSIC Rc 238


xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx

xxxxxx

xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx

xxx

xxx xxx



xxx

xxx xxx xxx xxxxxx


xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx

xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx

xxx xxx xxx xxx xxx xxx xxx xxx

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xxx xxx

xxxxxx

xxxxxx

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xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx

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xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx




xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxxxxx xxx xxx xxx xxx

INDEL22.2 Mb 22.8 Mb

c

Os09g20380 Os09g20400Os09g20390

DELETION

OsTPP7

17

Supplementary Figure 2 | Differences of qAG-9-2 region between KHO, IR64 and MSU7

and distribution of the OsTPP7 deletion across IRRI germplasm

a, Schematic of the qAG-9-2 region, assembled via a hybrid approach of Bacterial Artificial

Chromosome (BAC) deep sequencing and sequencing of overlapping amplicon clones. White

boxes on left and right indicate fine mapping flanking markers. Black boxes indicate gene

models (MSU ID) and grey arrows indicate direction of gene transcription. b, Mauve alignment

of the qAG-9-2 region from the Nipponbare reference assembly (MSU7), KHO and IR6411. First

row indicates size and relative position. Numbered rectangles indicate gene models (A-E =

LOC_Os09g20360 to LOC_Os09g20400 indicated in panel a). Green areas indicate well aligned

portions and grey areas indicate deletions. LOC_Os09g20390 encodes OsTPP7.

LOC_Os09g20380 encodes an unclassified transposable element that is partially deleted in IR64

and KHO. c, Schematic of the ~21 kb deletion region containing OsTPP7 as found in IR64.

Arrows indicate primer positions for co-dominant marker. Orange area indicates position within

deletion. d, Presence (white boxes) and absence (black boxes) of SNPseek37 core SNPs across

the larger qAG-9-2 region for 90 IRRI-derived varieties and breeding lines that are represented in

the 3000 genomes project12 (NSIC Rc222 and NSIC Rc238 are not present in SNPseek and were

added from IRRI-internal data). Each row represents a different genotype/breeding line (pink

lines indicating the subset named on the left) and each column a core SNP. The data shows

distribution of the OsTPP7-containing deletion across a representative range of IRRI-derived

cultivars including very early cultivars (IR6) and recently released varieties (NSIC Rc238).




18

a b

0

20

40

60

80

surv

ival

(%)

KHO IR64 NIL-AG1

a

b

c

0

40

60

Shoot Root

Leng

th (m

m)

IR64NIL-AG1KHO

***

***

******

***

***

20

c

Os09g20400 (E)Expressed protein

Os09g20360 (A)Expressed protein

Os09g20390 (D)OsTPP7

TUBULIN

Os09g20370 (B)Expressed protein

IR64 NIL-AG1 GC

AIR H2O AIR H2O

Days after seeding

0

10

20

30

40

0 2 3 4

IR64

NIL-AG1

KHO

Col

eopt

ilele

ngth

(mm

)

d

e

0

10

20

30

0 1 2 3 4

Days after seeding

IR64

NIL-AG1

KHO

α-am

ylas

e ac

tivity

(mm

olm

in-1

g-1pr

otei

n)

1

3

5

7

Moc

k

0.01 0.

1 1 2 3 4 5

T50

(day

)

ABA (µM)

IR64NIL-AG1

f

g h

- GA + GA - GA + GA

40

60

80

100

Mock PAC+0 PAC+0.1 PAC+10

T50

(hou

r)

GA (µM)

IR64NIL-AG1

IR64 NIL-AG10

10

20

30

40

Col

eopt

ilele

ngth

(mm

)

a b

c

d

19

Supplementary Figure 3 | qAG-9-2 and candidate gene analysis and qAG-9-2-dependent

AG-survival and seedling vigor phenotypes

a, Semi-quantitative RT-PCR for all genes in the qAG-9-2 candidate region performed with

mRNA obtained from IR64 and NIL-AG1 after four days of growth in the dark under air (AIR)

or submergence (H2O). α-tubulin (TUBULIN) served as a housekeeping control and genomic

DNA (GC) as a PCR control. b, Survival rates of the tolerant parent KHO, the susceptible parent

IR64 and NIL-AG1 after 21 days of growth under submergence (n = 24 ± s.e.m.), different

letters denote p<0.001. c, Means of coleoptile lengths after 2-4 days of growth in the dark under

submergence (DGDS) (n = 69, ± s.e.m., p<0.001 for all timepoints relative to IR64). d, Means

of α-amylase activity after 1-4 DGDS (n = 4, ± s.d., p<0.05 for all timepoints relative to IR64). e,

Root and shoot total length of IR64 (white bars), NIL-AG1 (light grey bars) and KHO (dark grey

bars) seedlings after four days of growth in the dark under aerobic conditions (n = 209, ± s.e.m.),

*** = p<0.001. f-g, Germination time (T50, time for half of all seeds to germinate) in the dark

under aerobic condition in the presence of GA plus 200 µM paclobutazol (PAC) (f) or ABA (g)

(n = 20). Mock=H20. h, Means of coleoptile lengths after 4 DGDS in the absence (- GA) and

presence (+ GA) of 10 µM GA3 (n = 132, ± s.e.m.) Different letters indicate differences with

p<0.001.




18

a b

0

20

40

60

80

surv

ival

(%)

KHO IR64 NIL-AG1

a

b

c

0

40

60

Shoot Root

Leng

th (m

m)

IR64NIL-AG1KHO

***

***

******

***

***

20

c

Os09g20400 (E)Expressed protein

Os09g20360 (A)Expressed protein

Os09g20390 (D)OsTPP7

TUBULIN

Os09g20370 (B)Expressed protein

IR64 NIL-AG1 GC

AIR H2O AIR H2O

Days after seeding

0

10

20

30

40

0 2 3 4

IR64

NIL-AG1

KHO

Col

eopt

ilele

ngth

(mm

)

d

e

0

10

20

30

0 1 2 3 4

Days after seeding

IR64

NIL-AG1

KHO

α-am

ylas

e ac

tivity

(mm

olm

in-1

g-1pr

otei

n)

1

3

5

7

Moc

k

0.01 0.

1 1 2 3 4 5

T50

(day

)

ABA (µM)

IR64NIL-AG1

f

g h

- GA + GA - GA + GA

40

60

80

100

Mock PAC+0 PAC+0.1 PAC+10

T50

(hou

r)

GA (µM)

IR64NIL-AG1

IR64 NIL-AG10

10

20

30

40

Col

eopt

ilele

ngth

(mm

)

a b

c

d

19

Supplementary Figure 3 | qAG-9-2 and candidate gene analysis and qAG-9-2-dependent

AG-survival and seedling vigor phenotypes

a, Semi-quantitative RT-PCR for all genes in the qAG-9-2 candidate region performed with

mRNA obtained from IR64 and NIL-AG1 after four days of growth in the dark under air (AIR)

or submergence (H2O). α-tubulin (TUBULIN) served as a housekeeping control and genomic

DNA (GC) as a PCR control. b, Survival rates of the tolerant parent KHO, the susceptible parent

IR64 and NIL-AG1 after 21 days of growth under submergence (n = 24 ± s.e.m.), different

letters denote p<0.001. c, Means of coleoptile lengths after 2-4 days of growth in the dark under

submergence (DGDS) (n = 69, ± s.e.m., p<0.001 for all timepoints relative to IR64). d, Means

of α-amylase activity after 1-4 DGDS (n = 4, ± s.d., p<0.05 for all timepoints relative to IR64). e,

Root and shoot total length of IR64 (white bars), NIL-AG1 (light grey bars) and KHO (dark grey

bars) seedlings after four days of growth in the dark under aerobic conditions (n = 209, ± s.e.m.),

*** = p<0.001. f-g, Germination time (T50, time for half of all seeds to germinate) in the dark

under aerobic condition in the presence of GA plus 200 µM paclobutazol (PAC) (f) or ABA (g)

(n = 20). Mock=H20. h, Means of coleoptile lengths after 4 DGDS in the absence (- GA) and

presence (+ GA) of 10 µM GA3 (n = 132, ± s.e.m.) Different letters indicate differences with

p<0.001.




20

Supplementary Figure 4 | Analysis of native promoter OsTPP7, OsTPP7 overexpression and

knock-out T-DNA insertion OsTPP7 transgenic lines

BLAST of TAIL-PCR amplicons for native promoter OsTPP7 lines AG1-1 (a) and AG1-2 (b),

showing sites of T-DNA insertions in the SALK genome browser (http://signal.salk.edu/cgi-

bin/RiceGE). Three primer homozygosity PCRs for AG1-1 (c) and AG1-2 (d) with two T-DNA

insertion flanking primers and one T-DNA left border primer as depicted (e). Lower bands

correspond to the mutant allele and upper bands correspond to the WT allele. NTC = no

b

c

Gene track

BAC track

cDNA track

TAIL track

d e

T-DN

A

f

a

OX

1 H

MO

X1

HW

OX

2 H

MO

X2

HW

AG

1-1

HM

AG

1-1

HW

AG

1-2

HM

AG

1-2

HW

AG

1-3

HM

AG

1-3

HW

NIL

-AG

1N

TC

Ladd

er

OsTPP7

TUBULIN

AG

1-1

HW

AG

1-1

HM

IR64

NTC

Ladd

er

AG

1-2

HW

AG

1-2

HM

IR64

NTC

Ladd

er

T-DNA

1 2

3

1 = PFG_TPP_F

2 = PFG_TPP_R

3 = TDNA-LB2

1-1

HWKOC

1-3

HMKO

1-2

HE

Ladd

er

DJCont

WTD

ongj

in

KO

KO

C

DN

A C

NTC

Ladd

er

OsTPP7

TUBULIN

g h iPFG_3A08739.L

Gene track

BAC track

cDNA track

TAIL track

21

template control. f, Semi-quantitative RT-PCR detection of OsTPP7 and α-tubulin (TUBULIN)

from mRNA obtained from embryos with coleoptiles after four days of growth in dark and under

submergence for 2 independent homozygous constitutive maize POLYUBIQUITIN promoter

(pZmUbi::OsTPP7; OX HM) lines and their respective null segregant (OX HW) lines, and 3

independent homozygous native promoter (pOsTPP7::OsTPP7; AG1 HM) lines and their

respective null segregants (AG1 HW) lines. Red box depicts lines used for further studies.

Under the observed conditions there were no obvious OsTPP7 transcript differences between

OX1, OX2, AG1-1 and AG1-2. The three bands for OsTPP7 correspond to three splice variants,

with splice variant 1 (LOC_Os09g20390.1) being predominant. g, Site of T-DNA insertion in

OsTPP7 exon 3 (CLON PFG_3A-08739.L) and primer positions for determination of

homozygosity. h, Demonstration of homozygosity by genomic PCR for T2 individuals of CLON

PFG_3A-08739.L in the Dongjin background. Bands in the upper panel correspond to the WT

allele amplified with the T-DNA flanking primers; bands in the lower panel correspond to the

mutant allele, amplified with one T-DNA flanking primer and one T-DNA left border primer.

KO=knockout T-DNA insertion line; DJ Cont=Dongjin control; HW=homozygous wild type

allele; HE=hemizygous allele; HM=homozygous mutant allele. i, Semi-quantitative RT-PCR for

OsTPP7 transcript in the Dongjin wildtype, the OsTPP7 T-DNA insertion homozygous mutant

(KO), the OsTPP7 T-DNA insertion null segregant (KOC) a Dongjin genomic control (DNA C)

and a no-template control (NTC). α-tubulin (TUBULIN) served as a housekeeping control.




20

Supplementary Figure 4 | Analysis of native promoter OsTPP7, OsTPP7 overexpression and

knock-out T-DNA insertion OsTPP7 transgenic lines

BLAST of TAIL-PCR amplicons for native promoter OsTPP7 lines AG1-1 (a) and AG1-2 (b),

showing sites of T-DNA insertions in the SALK genome browser (http://signal.salk.edu/cgi-

bin/RiceGE). Three primer homozygosity PCRs for AG1-1 (c) and AG1-2 (d) with two T-DNA

insertion flanking primers and one T-DNA left border primer as depicted (e). Lower bands

correspond to the mutant allele and upper bands correspond to the WT allele. NTC = no

b

c

Gene track

BAC track

cDNA track

TAIL track

d e

T-DN

A

f

a

OX

1 H

MO

X1

HW

OX

2 H

MO

X2

HW

AG

1-1

HM

AG

1-1

HW

AG

1-2

HM

AG

1-2

HW

AG

1-3

HM

AG

1-3

HW

NIL

-AG

1N

TC

Ladd

er

OsTPP7

TUBULIN

AG

1-1

HW

AG

1-1

HM

IR64

NTC

Ladd

er

AG

1-2

HW

AG

1-2

HM

IR64

NTC

Ladd

er

T-DNA

1 2

3

1 = PFG_TPP_F

2 = PFG_TPP_R

3 = TDNA-LB2

1-1

HWKOC

1-3

HMKO

1-2

HE

Ladd

er

DJCont

WT

Don

gjin

KO

KO

C

DN

A C

NTC

Ladd

er

OsTPP7

TUBULIN

g h iPFG_3A08739.L

Gene track

BAC track

cDNA track

TAIL track

21

template control. f, Semi-quantitative RT-PCR detection of OsTPP7 and α-tubulin (TUBULIN)

from mRNA obtained from embryos with coleoptiles after four days of growth in dark and under

submergence for 2 independent homozygous constitutive maize POLYUBIQUITIN promoter

(pZmUbi::OsTPP7; OX HM) lines and their respective null segregant (OX HW) lines, and 3

independent homozygous native promoter (pOsTPP7::OsTPP7; AG1 HM) lines and their

respective null segregants (AG1 HW) lines. Red box depicts lines used for further studies.

Under the observed conditions there were no obvious OsTPP7 transcript differences between

OX1, OX2, AG1-1 and AG1-2. The three bands for OsTPP7 correspond to three splice variants,

with splice variant 1 (LOC_Os09g20390.1) being predominant. g, Site of T-DNA insertion in

OsTPP7 exon 3 (CLON PFG_3A-08739.L) and primer positions for determination of

homozygosity. h, Demonstration of homozygosity by genomic PCR for T2 individuals of CLON

PFG_3A-08739.L in the Dongjin background. Bands in the upper panel correspond to the WT

allele amplified with the T-DNA flanking primers; bands in the lower panel correspond to the

mutant allele, amplified with one T-DNA flanking primer and one T-DNA left border primer.

KO=knockout T-DNA insertion line; DJ Cont=Dongjin control; HW=homozygous wild type

allele; HE=hemizygous allele; HM=homozygous mutant allele. i, Semi-quantitative RT-PCR for

OsTPP7 transcript in the Dongjin wildtype, the OsTPP7 T-DNA insertion homozygous mutant

(KO), the OsTPP7 T-DNA insertion null segregant (KOC) a Dongjin genomic control (DNA C)

and a no-template control (NTC). α-tubulin (TUBULIN) served as a housekeeping control.




22

0

30

60

90

120

150

0 0.2 0.4 0.6 0.8 1

Pho

spha

te p

rodu

ctio

n(n

mol

min

-1m

g-1pr

otei

n)

T6P (mM)

0

0.3

0.6

0.9

1.2

1.5

-0.2 0 0.2 0.4 0.6 0.8 1

[T6P

] V-1

T6P (mM)

OsTPP7

TEVprotease

TEVELU Lad

75 kD

50 kD

37 kD

25 kD

20 kD

Lad ELU CE+ CE-

a b

IR64 OX1 OX2e

Grown on 0.4 M glucose

Fres

h w

eigh

t (m

g)

0

40

80

120

160

IR64 OX1 OX2

0.4 M glucose

0.4 M sorbitol

***

f

dc

23

Supplementary Figure 5 | In vitro catalytic activity of E. coli expressed recombinant

OsTPP7 and high glucose sensitivity phenotype of OsTPP7 overexpression seedlings

a,b Coomassie blue stained SDS-PAGs with the final elution of Halo-Tag purified OsTPP7

(ELU), the TEV-protease used to cleave OsTPP7 from the resin-bound Halo-Tag (TEV), crude

protein extract from rhamnose-induced E. coli (CE+, arrow indicates induced OsTPP7), crude

protein extract from glucose inhibited culture (CE-) and the Precision Plus protein standard

(Lad). Apparent mass in kD of selected protein bands is indicated. Expected size for OsTPP7 is

41 kD (arrow). Expected size for TEV is 50 kD. c, Michaelis Menten kinetics for recombinant

OsTPP7, as monitored by phosphate release in relation to T6P concentration (n = 6, ± s.e.m.). d,

Hanes-Woolf plot of the same data set, suggesting an apparent Km = 0.2 mM by trend-line

regression. The calculated turnover rate (Kcat) was ~ 0.1/s. e, Phenotypes of IR64 and two

transgenic lines constitutively expressing OsTPP7 (OX) after 2 weeks of growth on plates

containing 0.4 M glucose. f, Total fresh weight of IR64 and two OX lines after 2 weeks of

growth on plates containing either 0.4 M glucose or 0.4 M sorbitol (n = 12 ± s.d., *** =

p<0.001).




22

0

30

60

90

120

150

0 0.2 0.4 0.6 0.8 1

Pho

spha

te p

rodu

ctio

n(n

mol

min

-1m

g-1pr

otei

n)

T6P (mM)

0

0.3

0.6

0.9

1.2

1.5

-0.2 0 0.2 0.4 0.6 0.8 1

[T6P

] V-1

T6P (mM)

OsTPP7

TEVprotease

TEVELU Lad

75 kD

50 kD

37 kD

25 kD

20 kD

Lad ELU CE+ CE-

a b

IR64 OX1 OX2e

Grown on 0.4 M glucose

Fres

h w

eigh

t (m

g)

0

40

80

120

160

IR64 OX1 OX2

0.4 M glucose

0.4 M sorbitol

***

f

dc

23

Supplementary Figure 5 | In vitro catalytic activity of E. coli expressed recombinant

OsTPP7 and high glucose sensitivity phenotype of OsTPP7 overexpression seedlings

a,b Coomassie blue stained SDS-PAGs with the final elution of Halo-Tag purified OsTPP7

(ELU), the TEV-protease used to cleave OsTPP7 from the resin-bound Halo-Tag (TEV), crude

protein extract from rhamnose-induced E. coli (CE+, arrow indicates induced OsTPP7), crude

protein extract from glucose inhibited culture (CE-) and the Precision Plus protein standard

(Lad). Apparent mass in kD of selected protein bands is indicated. Expected size for OsTPP7 is

41 kD (arrow). Expected size for TEV is 50 kD. c, Michaelis Menten kinetics for recombinant

OsTPP7, as monitored by phosphate release in relation to T6P concentration (n = 6, ± s.e.m.). d,

Hanes-Woolf plot of the same data set, suggesting an apparent Km = 0.2 mM by trend-line

regression. The calculated turnover rate (Kcat) was ~ 0.1/s. e, Phenotypes of IR64 and two

transgenic lines constitutively expressing OsTPP7 (OX) after 2 weeks of growth on plates

containing 0.4 M glucose. f, Total fresh weight of IR64 and two OX lines after 2 weeks of

growth on plates containing either 0.4 M glucose or 0.4 M sorbitol (n = 12 ± s.d., *** =

p<0.001).




24

OX1 OX2 KHO NIL-AG1 IR64

TUBULIN

OsTPP7

0

2

4

6

8

10

2 days

OsT

PP

7 ex

pres

sion

3 days 4 days

**

*a

0

2

4

6

8

10

AIR H2O

OsT

PP

7 ex

pres

sion

*b

c

0

0.5

1

1.5

2

2.5

3CIPK15

IR64

NIL66

0

1

2

3

4

Rel

ativ

e m

RN

A le

vel

OsTPP7

0

0.5

1

1.5

2

2.5

3

3.5SnRK1A

0

2

4

6

8

10

Rel

ativ

e m

RN

A le

vel

MYBS1

0

0.5

1

1.5

2

2.5EXP11

b

a a

a

a

b

a

a

bb

b

a

aa a

b

ab

ab

0

2

4

6

8ADH1

a a

b b

AIR H2O

AIR H2O AIR H2OAIR H2O

AIR H2O AIR H2O

d

0

1

2

3EXP3

abb

b

a

AIR H2O

0

400

800

1200

1600

AIR H2O

RAMY3D

a a

b b

AIR H2O0

0.5

1

1.5

2

2.5

Rel

ativ

e m

RN

A le

vels

GH17

ab

ab

b

a

AIR H2O

25

Supplementary Figure 6 | OsTPP7 transcript abundance between treatments and

quantitative PCR validation of RNAseq data on selected genes that are implicated in AG

OsTPP7 transcript levels determined by use of quantitative RT-PCR on RNA isolated from NIL-

AG1 embryos (dark grey bar) and coleoptiles (light grey bar) grown for 2-4 days in dark and

under submergence (H2O) or aerobic conditions (AIR) (a-c). a, OsTPP7 transcript abundance

(Expression) after 2-4 days under submergence is shown relative to the 2 day coleoptile-embryo

samples (white bar). b, Expression of OsTPP7 under submerged conditions relative to aerobic

conditions (white bars) in coleoptile-embryo samples after 2 days. POLYUBIQUTIN, UBIQITIN

and ACTIN served as references transcripts. Average fold changes ± standard errors as calculated

by REST software after 3000 iterations (n = 3), * = p<0.05. c, Semi-quantitative RT-PCR of

OsTPP7 transgene and α-TUBULIN (TUB) mRNA of 2 week old seedling leaves of two

independent transgenic IR64 lines constitutively expressing pZmUbi::OsTPP7 (OX1 and OX2),

KHO, NIL-AG1 and IR64. The transcript is absent in KHO and NIL-AG1, which contain native

OsTPP7 alleles, but present in OX lines which are constitutively expressing OsTPP7.

d, Determination of selected transcript levels by quantitative RT-PCR on RNA isolated from

IR64 (white bars) and NIL-AG1 (grey bars) embryo-coleoptile tissue of seedlings grown for 4

days in the dark and under submergence (H2O) or aerobic conditions (AIR). POLYUBIQUTIN

served as the reference transcript as levels of UBIQITIN and TUBULIN mRNAs were less

consistent. Data represent mean ± s.e.m. of four independent experiments (n=4). Significant

differences between genotypes and treatments were determined by ANOVA and are denoted by

different letters, p≤0.05.




24

OX1 OX2 KHO NIL-AG1 IR64

TUBULIN

OsTPP7

0

2

4

6

8

10

2 days

OsT

PP

7 ex

pres

sion

3 days 4 days

**

*a

0

2

4

6

8

10

AIR H2O

OsT

PP

7 ex

pres

sion

*b

c

0

0.5

1

1.5

2

2.5

3CIPK15

IR64

NIL66

0

1

2

3

4

Rel

ativ

e m

RN

A le

vel

OsTPP7

0

0.5

1

1.5

2

2.5

3

3.5SnRK1A

0

2

4

6

8

10

Rel

ativ

e m

RN

A le

vel

MYBS1

0

0.5

1

1.5

2

2.5EXP11

b

a a

a

a

b

a

a

bb

b

a

aa a

b

ab

ab

0

2

4

6

8ADH1

a a

b b

AIR H2O

AIR H2O AIR H2OAIR H2O

AIR H2O AIR H2O

d

0

1

2

3EXP3

abb

b

a

AIR H2O

0

400

800

1200

1600

AIR H2O

RAMY3D

a a

b b

AIR H2O0

0.5

1

1.5

2

2.5

Rel

ativ

e m

RN

A le

vels

GH17

ab

ab

b

a

AIR H2O

25

Supplementary Figure 6 | OsTPP7 transcript abundance between treatments and

quantitative PCR validation of RNAseq data on selected genes that are implicated in AG

OsTPP7 transcript levels determined by use of quantitative RT-PCR on RNA isolated from NIL-

AG1 embryos (dark grey bar) and coleoptiles (light grey bar) grown for 2-4 days in dark and

under submergence (H2O) or aerobic conditions (AIR) (a-c). a, OsTPP7 transcript abundance

(Expression) after 2-4 days under submergence is shown relative to the 2 day coleoptile-embryo

samples (white bar). b, Expression of OsTPP7 under submerged conditions relative to aerobic

conditions (white bars) in coleoptile-embryo samples after 2 days. POLYUBIQUTIN, UBIQITIN

and ACTIN served as references transcripts. Average fold changes ± standard errors as calculated

by REST software after 3000 iterations (n = 3), * = p<0.05. c, Semi-quantitative RT-PCR of

OsTPP7 transgene and α-TUBULIN (TUB) mRNA of 2 week old seedling leaves of two

independent transgenic IR64 lines constitutively expressing pZmUbi::OsTPP7 (OX1 and OX2),

KHO, NIL-AG1 and IR64. The transcript is absent in KHO and NIL-AG1, which contain native

OsTPP7 alleles, but present in OX lines which are constitutively expressing OsTPP7.

d, Determination of selected transcript levels by quantitative RT-PCR on RNA isolated from

IR64 (white bars) and NIL-AG1 (grey bars) embryo-coleoptile tissue of seedlings grown for 4

days in the dark and under submergence (H2O) or aerobic conditions (AIR). POLYUBIQUTIN

served as the reference transcript as levels of UBIQITIN and TUBULIN mRNAs were less

consistent. Data represent mean ± s.e.m. of four independent experiments (n=4). Significant

differences between genotypes and treatments were determined by ANOVA and are denoted by

different letters, p≤0.05.




26

OsTPP7 = LOC_Os09g20390, CIPK15 = LOC_Os11g02240 (upstream regulator of SnRK119),

SNRK1A = LOC_Os05g45420 (global integrator of energy signaling28-30), MYBS1 =

LOC_Os01g34060 (α-amylase-activating transcription factor20, regulated by SnRK129), EXP3 =

LOC_Os10g40720 and EXP11 = LOC_Os02g44108 (expansins, proposed to be involved in cell

elongation20), GH17 = LOC_Os01g71860 (Family 17 hydrolase, proposed to be involved in cell

wall loosening), ADH1 = LOC_Os11g10480 (alcohol dehydrogenase 1, key fermentative

enzyme20, involved in hypoxia tolerance), RAmy3D = LOC_Os08g36910 (α-amylase shown to

be involved in hypoxia tolerance20)

27

Starch

Maltose

NIL-‐AG1

T6PSnRK1TPP

Endosperm EmbryoScutellum

Starch

Maltose

α-‐amylases

IR64

T6P TrehaloseSnRK1TPP

HexosesSucrose

T6P/Sucrose ColeoptileMYBS1

α-‐amylases (3)

Sucrose (5) Hexoses (6)

Trehalose (1)

Elongation Growth (2)

MYBS1 (4)

OsTPP7

T6P/Sucrose




26

OsTPP7 = LOC_Os09g20390, CIPK15 = LOC_Os11g02240 (upstream regulator of SnRK119),

SNRK1A = LOC_Os05g45420 (global integrator of energy signaling28-30), MYBS1 =

LOC_Os01g34060 (α-amylase-activating transcription factor20, regulated by SnRK129), EXP3 =

LOC_Os10g40720 and EXP11 = LOC_Os02g44108 (expansins, proposed to be involved in cell

elongation20), GH17 = LOC_Os01g71860 (Family 17 hydrolase, proposed to be involved in cell

wall loosening), ADH1 = LOC_Os11g10480 (alcohol dehydrogenase 1, key fermentative

enzyme20, involved in hypoxia tolerance), RAmy3D = LOC_Os08g36910 (α-amylase shown to

be involved in hypoxia tolerance20)

27

Starch

Maltose

NIL-‐AG1

T6PSnRK1TPP

Endosperm EmbryoScutellum

Starch

Maltose

α-‐amylases

IR64

T6P TrehaloseSnRK1TPP

HexosesSucrose

T6P/Sucrose ColeoptileMYBS1

α-‐amylases (3)

Sucrose (5) Hexoses (6)

Trehalose (1)

Elongation Growth (2)

MYBS1 (4)

OsTPP7

T6P/Sucrose




28

Supplementary Figure 7 | Proposed mechanism of OsTPP7 function in enhancing anaerobic

germination

Blue ovals indicate upregulated metabolites or transcripts associated with presence of OsTPP7

(green oval). Orange oval indicated downregulated metabolite ratio associated with presence of

OsTPP7. Solid arrows indicate steps or interactions supported by the literature. Dashed arrows

indicate interactions for which there is no experimental evidence in the literature. White tringles

indicate flux of sugars. Bold outlines indicate increased flux.

In the OsTPP7-lacking IR64 background carbon fluxes from starch to hexoses for fermentative

energy production and supply of carbon for growth related macromolecules is limited, which is

at least in part due feedback inhibition of starch mobilization in source by sucrose levels in

sink17,19. This is assumed to be partially mediated through SnRK1 and T6P levels9, 29. T6P

contents are tightly correlated to sucrose contents22, resulting in stable T6P:sucrose ratios25.

In the OsTPP7-containing NIL-AG1 and AG1-1 lines, T6P conversion to trehalose is enhanced

as evident from higher trehalose (1) contents (Fig. 2c). This is speculated to partially alleviate

feedback inhibition of sugar fluxes from source to sink, allowing for increased carbon utilization

for energy and macromolecule production, ultimately enhancing coleoptile elongation (2) (Fig.

1b and c, Supplementary Fig. 3c).

Higher α-amylase activities (3) (Fig. 1c, Supplementary Fig. 3d) due to increased MYBS1

expression (4) (Supplementary Fig. 6d) lead to higher sucrose (5) (Fig. 2c) and hexose (6) (Fig.

2d) contents in the embryo-coleoptile. Higher sucrose contents are thought to translate to

moreT6P25, which we speculated to be buffered by T6P breakdown via OsTPP7 (Supplementary

Fig. 5) leading to T6P contents similar to those observed in IR64 (Fig 2a).

29

IR64NIL-AG1

IR42PSB Rc82

NSIC Rc222KHO

FR13A

a

IR42

IR64-AG1

FR13A

IR64

c

b




28

Supplementary Figure 7 | Proposed mechanism of OsTPP7 function in enhancing anaerobic

germination

Blue ovals indicate upregulated metabolites or transcripts associated with presence of OsTPP7

(green oval). Orange oval indicated downregulated metabolite ratio associated with presence of

OsTPP7. Solid arrows indicate steps or interactions supported by the literature. Dashed arrows

indicate interactions for which there is no experimental evidence in the literature. White tringles

indicate flux of sugars. Bold outlines indicate increased flux.

In the OsTPP7-lacking IR64 background carbon fluxes from starch to hexoses for fermentative

energy production and supply of carbon for growth related macromolecules is limited, which is

at least in part due feedback inhibition of starch mobilization in source by sucrose levels in

sink17,19. This is assumed to be partially mediated through SnRK1 and T6P levels9, 29. T6P

contents are tightly correlated to sucrose contents22, resulting in stable T6P:sucrose ratios25.

In the OsTPP7-containing NIL-AG1 and AG1-1 lines, T6P conversion to trehalose is enhanced

as evident from higher trehalose (1) contents (Fig. 2c). This is speculated to partially alleviate

feedback inhibition of sugar fluxes from source to sink, allowing for increased carbon utilization

for energy and macromolecule production, ultimately enhancing coleoptile elongation (2) (Fig.

1b and c, Supplementary Fig. 3c).

Higher α-amylase activities (3) (Fig. 1c, Supplementary Fig. 3d) due to increased MYBS1

expression (4) (Supplementary Fig. 6d) lead to higher sucrose (5) (Fig. 2c) and hexose (6) (Fig.

2d) contents in the embryo-coleoptile. Higher sucrose contents are thought to translate to

moreT6P25, which we speculated to be buffered by T6P breakdown via OsTPP7 (Supplementary

Fig. 5) leading to T6P contents similar to those observed in IR64 (Fig 2a).

29

IR64NIL-AG1

IR42PSB Rc82

NSIC Rc222KHO

FR13A

a

IR42

IR64-AG1

FR13A

IR64

c

b




30

Supplementary Figure 8 | Genotype and performance of NIL-AG1 under direct seeding

Pan-genome similarities of the tested lines to IR64 determined using 4037 single nucleotide

polymorphism (SNP) markers across the genome are: 99.0% for NIL-AG1, 89.7% for IR42,

87.8% for PSB Rc82, 85.3% for NSIC Rc222, 77.5% for KHO, and 67.5% for FR13A. a,

Graphical genotype of 10.6-13.7 Mb region of chromosome 9. A single SNP at 12.33 Mb

between the KHO allele in NIL-AG1 at the qAG-9-2 locus is indicated in red. b, Performance of

NIL-AG1 compared to the original parent IR64, donor KHO, and FR13A under AG stress with

dry seeds sown underwater in the greenhouse. c, Comparison of direct seeded IR64, IR42 and

FR13A under AG stress in the field condition.

31

Supplementary Table 1| Gene ontology enrichment (GO) analysis of differentially

regulated genes between a native promoter Os TPP7 transgenic line (AG1-1) and the

corresponding null segregant line (entries can be found in Supplementary Table 3)

GO analyzed with AgriGO: http://bioinfo.cau.edu.cn/agriGO/Most significantly differentially up regulated genes (FDR <0.05) (n=62); too few down regualted genes for GOGO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0005618 C cell wall 6 31 1179 34296 0.00059 0.018GO:0030312 C external encapsulating structure 6 31 1189 34296 0.00061 0.018GO:0008361 P regulation of cell size 3 31 567 34296 0.014 0.36GO:0016049 P cell growth 3 31 567 34296 0.014 0.36GO:0090066 P regulation of anatomical structure size 3 31 567 34296 0.014 0.36GO:0065008 P regulation of biological quality 4 31 863 34296 0.0074 0.36GO:0032535 P regulation of cellular component size 3 31 567 34296 0.014 0.36GO:0005576 C extracellular region 3 31 730 34296 0.028 0.55GO:0040007 P growth 3 31 736 34296 0.029 0.59GO:0005975 P carbohydrate metabolic process 4 31 1439 34296 0.04 0.71GO:0008289 F lipid binding 2 31 346 34296 0.039 0.71

Most Significantly differentially up regulated genes (|FC| > 2, p < 0.05 (n=192)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0030312 C external encapsulating structure 22 166 1189 34296 9.90E-‐08 6.40E-‐06GO:0005618 C cell wall 22 166 1179 34296 8.50E-‐08 6.40E-‐06GO:0006950 P response to stress 50 166 4660 34296 3.00E-‐08 8.80E-‐06GO:0050896 P response to stimulus 60 166 6928 34296 1.50E-‐06 0.00023GO:0009628 P response to abiotic stimulus 30 166 3022 34296 0.00013 0.013GO:0015979 P photosynthesis 8 166 324 34296 0.00022 0.016GO:0009607 P response to biotic stimulus 17 166 1404 34296 0.00053 0.032GO:0005773 C vacuole 18 166 1723 34296 0.0019 0.083GO:0005488 F binding 91 166 15115 34296 0.0035 0.21

Most Significantly differentially down regulated genes (|FC| > 2, p < 0.05 (n=80)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0016787 F hydrolase activity 17 58 4293 34296 0.00055 0.015GO:0003824 F catalytic activity 35 58 13508 34296 0.001 0.015GO:0005623 C cell 50 58 22048 34296 0.00018 0.016GO:0005102 F receptor binding 2 58 69 34296 0.0065 0.063GO:0009991 P response to extracellular stimulus 5 58 393 34296 0.00056 0.099GO:0007154 P cell communication 5 58 512 34296 0.0018 0.16GO:0008289 F lipid binding 3 58 346 34296 0.021 0.16GO:0006950 P response to stress 16 58 4660 34296 0.0038 0.19GO:0009628 P response to abiotic stimulus 12 58 3022 34296 0.0042 0.19GO:0009605 P response to external stimulus 5 58 681 34296 0.006 0.21GO:0050896 P response to stimulus 20 58 6928 34296 0.008 0.23GO:0005773 C vacuole 8 58 1723 34296 0.0083 0.38GO:0005829 C cytosol 11 58 3345 34296 0.023 0.7




30

Supplementary Figure 8 | Genotype and performance of NIL-AG1 under direct seeding

Pan-genome similarities of the tested lines to IR64 determined using 4037 single nucleotide

polymorphism (SNP) markers across the genome are: 99.0% for NIL-AG1, 89.7% for IR42,

87.8% for PSB Rc82, 85.3% for NSIC Rc222, 77.5% for KHO, and 67.5% for FR13A. a,

Graphical genotype of 10.6-13.7 Mb region of chromosome 9. A single SNP at 12.33 Mb

between the KHO allele in NIL-AG1 at the qAG-9-2 locus is indicated in red. b, Performance of

NIL-AG1 compared to the original parent IR64, donor KHO, and FR13A under AG stress with

dry seeds sown underwater in the greenhouse. c, Comparison of direct seeded IR64, IR42 and

FR13A under AG stress in the field condition.

31

Supplementary Table 1| Gene ontology enrichment (GO) analysis of differentially

regulated genes between a native promoter Os TPP7 transgenic line (AG1-1) and the

corresponding null segregant line (entries can be found in Supplementary Table 3)

GO analyzed with AgriGO: http://bioinfo.cau.edu.cn/agriGO/Most significantly differentially up regulated genes (FDR <0.05) (n=62); too few down regualted genes for GOGO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0005618 C cell wall 6 31 1179 34296 0.00059 0.018GO:0030312 C external encapsulating structure 6 31 1189 34296 0.00061 0.018GO:0008361 P regulation of cell size 3 31 567 34296 0.014 0.36GO:0016049 P cell growth 3 31 567 34296 0.014 0.36GO:0090066 P regulation of anatomical structure size 3 31 567 34296 0.014 0.36GO:0065008 P regulation of biological quality 4 31 863 34296 0.0074 0.36GO:0032535 P regulation of cellular component size 3 31 567 34296 0.014 0.36GO:0005576 C extracellular region 3 31 730 34296 0.028 0.55GO:0040007 P growth 3 31 736 34296 0.029 0.59GO:0005975 P carbohydrate metabolic process 4 31 1439 34296 0.04 0.71GO:0008289 F lipid binding 2 31 346 34296 0.039 0.71

Most Significantly differentially up regulated genes (|FC| > 2, p < 0.05 (n=192)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0030312 C external encapsulating structure 22 166 1189 34296 9.90E-‐08 6.40E-‐06GO:0005618 C cell wall 22 166 1179 34296 8.50E-‐08 6.40E-‐06GO:0006950 P response to stress 50 166 4660 34296 3.00E-‐08 8.80E-‐06GO:0050896 P response to stimulus 60 166 6928 34296 1.50E-‐06 0.00023GO:0009628 P response to abiotic stimulus 30 166 3022 34296 0.00013 0.013GO:0015979 P photosynthesis 8 166 324 34296 0.00022 0.016GO:0009607 P response to biotic stimulus 17 166 1404 34296 0.00053 0.032GO:0005773 C vacuole 18 166 1723 34296 0.0019 0.083GO:0005488 F binding 91 166 15115 34296 0.0035 0.21

Most Significantly differentially down regulated genes (|FC| > 2, p < 0.05 (n=80)GO_acc term_type Term queryitem querytotal bgitem bgtotal pvalue FDRGO:0016787 F hydrolase activity 17 58 4293 34296 0.00055 0.015GO:0003824 F catalytic activity 35 58 13508 34296 0.001 0.015GO:0005623 C cell 50 58 22048 34296 0.00018 0.016GO:0005102 F receptor binding 2 58 69 34296 0.0065 0.063GO:0009991 P response to extracellular stimulus 5 58 393 34296 0.00056 0.099GO:0007154 P cell communication 5 58 512 34296 0.0018 0.16GO:0008289 F lipid binding 3 58 346 34296 0.021 0.16GO:0006950 P response to stress 16 58 4660 34296 0.0038 0.19GO:0009628 P response to abiotic stimulus 12 58 3022 34296 0.0042 0.19GO:0009605 P response to external stimulus 5 58 681 34296 0.006 0.21GO:0050896 P response to stimulus 20 58 6928 34296 0.008 0.23GO:0005773 C vacuole 8 58 1723 34296 0.0083 0.38GO:0005829 C cytosol 11 58 3345 34296 0.023 0.7




32

Supplementary Table 2| Grain quality parameters of NIL-AG1 compared to IR64

Entries Length

(mm)

Width

(mm)

Chalkiness

(%)

ACa

(%)

GT GC

(mm)

BR

(%)

MR

(%)

HR

(%)

IR64 6.95 2.27 1.9 24.3 I 78 78.9 70.3 64.3

NIL-AG1 6.96 2.33 2.5 26.1 I 80 79.3 70.4 63.0

aAC=amylose content, GT=gelatinization temperature, GC=gel consistency, BR=brown rice,

MR=milled rice, HR=head rice, I=intermediate

Supplementary Table 3| RNA-seq analysis of the AG1-1 trasngenic (TPP7::TPP7 in IR64)

and IR64 coleoptile-embryo tissue after 4 d germination in darkness

Supplementary Table 4| List of primers

33

Supplementary References

41. Ammiraju, J. S. S. et al. The Oryza bacterial artificial chromosome library resource:

construction and analysis of 12 deep-coverage large-insert BAC libraries that represent

the 10 genome types of the genus Oryza. Genome Res. 16, 140-147 (2006).

42. Church, G. M. & Gilbert, W. Genomic sequencing. Proceedings of the National Academy

of Sciences 81, 1991-1995 (1984).

43. Liu, Y. G., Mitsukawa, N., Oosumi, T. & Whittier, R. F. Efficient isolation and mapping

of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.

Plant J. 8, 457-463 (1995).

44. Cervera, M. in Transgenic Plants: Methods and Protocols Vol. 286 Methods in

Molecular Biology (ed Leandro PeÃ±a) 203-213 (Humana Press, 2004).

45. Fukao, T., Yeung, E. & Bailey-Serres, J. The submergence tolerance gene SUB1A delays

leaf senescence under prolonged darkness through hormonal regulation in rice. Plant

Physiol. 160, 1795-1807 (2012).

46. Pfaffl, M. W., Horgan, G. W. & Dempfle, L. Relative expression software tool (REST)

for group-wise comparison and statistical analysis of relative expression results in real-

time PCR. Nucleic Acids Res. 30, e36 (2002).

47. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-

time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408

(2001).

48. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T)

method. Nat Protoc 3, 1101-1108 (2008).




32

Supplementary Table 2| Grain quality parameters of NIL-AG1 compared to IR64

Entries Length

(mm)

Width

(mm)

Chalkiness

(%)

ACa

(%)

GT GC

(mm)

BR

(%)

MR

(%)

HR

(%)

IR64 6.95 2.27 1.9 24.3 I 78 78.9 70.3 64.3

NIL-AG1 6.96 2.33 2.5 26.1 I 80 79.3 70.4 63.0

aAC=amylose content, GT=gelatinization temperature, GC=gel consistency, BR=brown rice,

MR=milled rice, HR=head rice, I=intermediate

Supplementary Table 3| RNA-seq analysis of the AG1-1 trasngenic (TPP7::TPP7 in IR64)

and IR64 coleoptile-embryo tissue after 4 d germination in darkness

Supplementary Table 4| List of primers

33

Supplementary References

41. Ammiraju, J. S. S. et al. The Oryza bacterial artificial chromosome library resource:

construction and analysis of 12 deep-coverage large-insert BAC libraries that represent

the 10 genome types of the genus Oryza. Genome Res. 16, 140-147 (2006).

42. Church, G. M. & Gilbert, W. Genomic sequencing. Proceedings of the National Academy

of Sciences 81, 1991-1995 (1984).

43. Liu, Y. G., Mitsukawa, N., Oosumi, T. & Whittier, R. F. Efficient isolation and mapping

of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.

Plant J. 8, 457-463 (1995).

44. Cervera, M. in Transgenic Plants: Methods and Protocols Vol. 286 Methods in

Molecular Biology (ed Leandro PeÃ±a) 203-213 (Humana Press, 2004).

45. Fukao, T., Yeung, E. & Bailey-Serres, J. The submergence tolerance gene SUB1A delays

leaf senescence under prolonged darkness through hormonal regulation in rice. Plant

Physiol. 160, 1795-1807 (2012).

46. Pfaffl, M. W., Horgan, G. W. & Dempfle, L. Relative expression software tool (REST)

for group-wise comparison and statistical analysis of relative expression results in real-

time PCR. Nucleic Acids Res. 30, e36 (2002).

47. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-

time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408

(2001).

48. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T)

method. Nat Protoc 3, 1101-1108 (2008).




34

49. Mustroph, A. et al. Profiling translatomes of discrete cell populations resolves altered

cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 106, 18843-

18848 (2009).

50. Horan, K. et al. Annotating genes of known and unknown function by large-scale

coexpression analysis. Plant Physiol.147, 41-57 (2008).


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SUPPLEMENTARY INFORMATION · 2015-09-23 · SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.124 ... Hlan On (KHO) in the QTL region, while selecting against the rest of the genome